Anticancer, anti-inflammatory and analgesic activities of aminoalcohol-based quinoxaline small molecules

ABSTRACT Purpose: Bioactive molecules are relevant to fight cancer and associated conditions. Quinoxaline is a privileged N-heterocycle, notably as anticancer agents. Herein, we report the evaluation of the quinoxaline derivatives DEQX and OAQX as anticancer agents, as well as in function of their anti-inflammatory and analgesic activities. Methods: Quinoxalines were synthesized and tested as anticancer agents based on cell viability and Annexin V-FITC apoptosis. Anti-inflammatory activity was evaluated from mouse carrageenan peritonitis and levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-alfa for enzyme-linked immunosorbent assay. Hot-plate and acetic acid-induced writing test were employed to investigate analgesia. Results: Both reduced the Ht-29 cell viability in a dependent-concentration manner (p < 0.001). Total apoptosis was detected for cells treated with 12.5 and 25 µg/mL of both the compounds for 24 and 48 h (all doses, p < 0.0001). DEQX (all doses, p < 0.01) and OAQX (all doses, p < 0.001) acted in leukocyte migration and decreased the IL-1β and TNF-β levels (p < 0.05). DEQX (all doses, p < 0.05) and OAQX (5mg/kg, p < 0.001) showed peripheral analgesic effect. Conclusions: In-vitro and in-vivo results suggest that these quinoxalines are promising for application in pharmacological area due to their anticancer, anti-inflammatory, and peripheric analgesia.


Introduction
Cancer is one of the most common and deadly non-communicable diseases of the 21 st century, and intensive efforts have been devoted to researching new therapeutic products 1 .Nowadays, with the advances in medicinal chemistry, targeted strategies have emerged in the last decades as promising solutions to overcome the challenges of oncology treatments [2][3][4][5][6] .Apoptosis resistance is one of the most important hallmarks of cancer, and many investigations have Anticancer, anti-inflammatory and analgesic activities of aminoalcohol-based quinoxaline small molecules indicated that mitochondrial dysfunction is involved in this process [7][8][9][10][11][12] .Phosphatidylserine (PS) is a phospholipid naturally present in the cellular membrane of healthy cells; when exposed to an external leaflet of the plasma membrane, it can act as apoptosis signaling to the immune system 13 .Therefore, the annexin V binding assay is directly related to cancer studies 14,15 .
Nitrogen heterocycles are key constituents in many biological reactions and synthetic bioactive molecules, and these features have driven the advances in drug development 16 .Quinoxaline is solidified as a privileged N-heterocycle in biological fields, especially in medicinal chemistry, due to its presence in many bioactive structures against different pathologies, notably as anticancer agents based on their in-vitro and in-vivo activities, as well as in-silico studies, and involving different mechanisms of action [17][18][19][20][21][22][23][24][25][26][27][28][29] .
Based on the role of simple quinoxaline derivatives as a promissing target for cancer treatment, this paper reports anticancer activity of two quinoxaline derivatives, DEQX and OAQX (Fig. 1), which were previously considered as anticancer agent, but they have never been tested for biological purposes of any nature 23 .The in-vitro antitumor activity of DEQX and OAQX was evaluated by cell viability using a colorectal cancer cell line (Ht-29), and annexin V and propidium iodide labeling were employed to evaluate cell death.In addition, as inflammatory responses are indicated as a quite relevant factor in different stages of tumor development 30,31 , the effect of both quinoxaline derivatives on mouse carrageenan peritonitis and levels of pro-inflammatory interleukin (IL)-1 β and tumor necrosis factor (TNF)-α were evaluated.Lastly, the central and peripheral analgesic activities of DEQX and OAQX were investigated.

Synthesis of quinoxaline derivatives
Compounds DEQX and OAQX were synthesized according previously reported, and spectroscopic data are coherent to their molecular structures 23 .

In-vitro antitumor activity
The following reagents were purchased as indicated: Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Grand Island, NY, United States of America), 10% (v/v) heat-inactivated fetal bovine serum (CULTILAB LTDA/Brazil), trypsin/EDTA (Gibco BRL, Life Technologies, Grand Island, NY, United States of America), and cisplatin (citoplax, 50 mg, Bergamo Taboão da Serra, SP, Brazil).A colorectal cancer cell line (Ht-29) was purchased from the Culture Collection of the Universidade Federal do Rio de Janeiro (RJCB Collection, Rio de Janeiro, RJ, Brazil).Ht-29 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum.Molecules of DEQX and OAQX were diluted in DMSO (1%) and cisplatin diluted in medium (DMEM).All the solutions were filtered using a 0.22-mm minipore membrane, then were aliquoted and stored at -20 °C.

Cell viability
Due to the different sensitivity of cancer cells (1 × 105) to the molecules DEQX and OAQX, the optimal exposure time for each cell line was determined in a pilot study in order to obtain a dose-dependent effect.The DMSO (1%) vehicle of DEQX and OAQX was also tested.Viability was determined at 24 h for Ht-29, and different concentrations (3.125-200 μg/mL in aqueous suspensions) of molecules were placed into the wells.Cell viability was determined by trypan blue exclusion assay.Briefly, cell aliquots were mixed with same volume of 0.5% (w/v) trypan blue and incubated at room temperature for 5 minutes.The number of viable cells was calculated using a hemocytometer.Data was analyzed using GraphPad 5.0 (Prism Software, United States of America) to determine interval of confidence of 50 (IC50).

Annexin V and propidium iodide staining
Cells were plated in 6-well plates (5 × 10 5 cells/well) with 2 mL medium/well (triplicate).After 24 h, concentrations of both the compounds (3.125, 6.25, and 12.5 µg/mL) and cisplatin (50 and 100 µM) were added for 24 and 48 h.In parallel, control cells were maintained in culture medium without molecules and cisplatin.The cells were then assayed using the annexin V-FITC apoptosis detection kit I (Biosciences Pharmingen, San Diego, CA, United States of America).Annexin V-FITC and propidium iodide (PI) were added to the cellular suspension according to the manufacturer's instructions.Each sample of cell line were then analyzed using a FACS Calibur cytometer (BD Bioscience, Franklin Lakes, NJ, United States of America) and FlowJo software (BD Biosciences).Annexin VFITC-positive/PI-negative cells were identified as cells in the early stages of apoptosis, while annexin V-FITC-positive/PI-positive cells were identified as cells in the late stages of apoptosis, or cells that were undergoing necrosis.

Animals
Male and female Swiss mice (25-35 g) and rats (200-250 g), respectively, were obtained from Bioterio of the Biophysical and Pharmacology Department.All animals were housed in an animal room under standard laboratory conditions of 22 ± 2 °C and 12-h light/12-h dark cycle and fed with pellet food and water ad libitum.They were acclimatized for seven days before the experiments started and fasted for 12 h prior to the experiments.Animal welfare and experimental procedures were in strict accordance with Ethics Committee on Animal Use, approved protocol no.047/2013 and no.UFRN 047/2013.

Mouse carrageenan peritonitis
The female rats were divided into eight groups (n = 6/group).Compounds of DEQX and OAQX were administered orally at doses of 0.5, 1 and 5 mg/kg.Positive control group was vehicle (10 mL/kg, p.o.).Indomethacin (10 mg/kg; p.o.) was included as a standard group.Carrageenan (Sigma-Aldrich) (0.25 mL, 1% in saline) was intraperitoneally injected 30 min later the treatment with compounds, vehicle and indomethacin, and after 4 h the animals were sacrificed by thiopental (100 mg/kg) for further investigation.The total leukocyte count was determined in a Neubauer chamber 32 .

Hot-plate test: central analgesic activity
The hot-plate test was carried out using a hot-plate apparatus (model Insight, São Paulo, SP, Brazil), maintained at 55 ± 0.5 °C.The male Swiss mice were divided into five groups of six animals each and were fasted overnight.Only Anticancer, anti-inflammatory and analgesic activities of aminoalcohol-based quinoxaline small molecules mice that showed initial nociceptive responses (licking of the forepaws or jumping) between 3 and 19 were used for additional experiments.The chosen mice were pre-treated with compounds of DEQX and OAQX (0.5, 1 and 5 mg/kg; p.o.), and 30 min later the measurements were taken.Positive control group was treatment with vehicle (10 mL/kg, p.o.).A morphine group (10 mg/kg; i.p.) was included as a standard group.The cut-off time was set at 30 seconds to minimize skin damage.The reaction time, i.e., the amount of time it takes the animal to lick their forepaws or jump off its hot plate, was measured at 0, 30, 60, 90 and 120 min after the administration 35 .After, the animals were sacrificed by thiopental (100 mg/kg).

Acetic acid-induced abdominal writhing test: peripheral analgesic activity
The method of Koster et al. was used for this test 36 .The female Swiss mice (seven for group) were divided into five groups of six mice each and fasted overnight.The animals were treated with indomethacin (standard group, 10 mg/kg, p.o.), vehicle (10 ml/kg, p.o.) and compounds of DEQX and OAQX (0.5, 1 and 5 mg/kg, p.o.).The mice were treated with acetic acid (0.6%, v/v in saline, 10 mL/kg, i.p.) 30 minutes after the already-mentioned treatment was carried out.The number of writhes was counted for 20 min.Afterwards, the animals were sacrificed by thiopental (100 mg/kg).

Experimental outcomes
The animals were paired for sex, weight, age.During treatment, all groups were evaluated coat, motor activity (open Field) and death.No changes were recorded between groups.

Statistical analysis
All experiments were performed at least in triplicate, and significant differences between groups were calculated using analysis of variance (ANOVA) and Bonferroni's test, as indicated.P < 0.05 was considered statistically significant.

Chemistry
Quinoxaline derivatives DEQX and OAQX were obtained by reaction of previously prepared 2,3-dichloroquinoxaline (DCQX) and appropriate aminoalcohols, ethanolamine and diethanolamine, respectively, under heating for 3 hours (Fig. 1).The target compounds were obtained in good yielding, 82-87%, and infrared (IR), as well as 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopies data, is in agreement with those reported in literature 23 .
Source: Elaborated by the authors.

Cytotoxicity
Ht-29 cells showed a decreased growth when they were submitted to lower concentrations of DEQX and OAQX.
According to triplan blue assay results, the concentrations of DEQX and OAQX significantly affected on cell viability in a concentration-dependent manner (3.125-200 µg/mL).As presented in Figs.2a-b, the lowest mortality rate was obtained for DEQX and OAQX at concentrations of 3.125 (p > 0.0001), whereas the highest mortality rate was obtained at 200 µg/mL (p < 0.0001).In addition, DEQX and OAQX were cytotoxic on Ht-29 cells with a half-maximal inhibiting concentration value (IC50) of 12.5 µg/mL, (p < 0.05) (Fig. 2c).However, the concentrations 6.For Ht-29 cells, Figures 4 (a-e) and 5 (a-e) show control group treatment with 6.25, 12.5, and 25 µg/mL of DEQX compound induced early and late apoptosis either to 24 or 48 h after treatment (Figs. 3a and 4b-g).When Ht-29 cells were treated with 6.25, 12.5, and 25 µg/mL by OAQX compound, it also induced early and late apoptosis both 24 and 48 h after treatment (Fig. 3b and 5b-5g).Ht-29 cells were treated with Ht-29 cell (50 and 100 µM), and both the early and late stage apoptosis were detected 24 and 48 h after treatment.However, a higher percentage of cells in the late stages of apoptosis were observed (Fig. 3, 4i-j and 5i-j).Comp-FITC-A 0 10 2 10 3 10 4     Source: Elaborated by the authors.Anticancer, anti-inflammatory and analgesic activities of aminoalcohol-based quinoxaline small molecules

Anti-inflammatory effect in model carrageenan-induced peritonitis
In order to evaluate a possible inhibitory effect of DEQX (p < 0.01) and OAQX (p < 0.001), the carrageenan-induced peritonitis test was used on all doses on cell recruitment into the peritoneal cavity (Fig. 6).The negative control group showed an increase in the numbers of leukocytes from peritoneal exudates, and indomethacin showed an inhibitory effect on cell recruitment into the peritoneal cavity (p < 0.001).
Source: Elaborated by the authors.

Effects on inflammatory activity levels of interleukin-1β and tumor necrosis factor-α
The group of DEQX decreased levels of IL-1β (0.5 and 1 mg/kg, p < 0.001; and 5 mg/kg, p < 0.05) compared to positive control (Fig. 7).Levels of anti-inflammatory cytokine IL-1β were decreased in the group treated with 0.5 and 5 mg/ kg of OAQX compared to positive control (p < 0.001).Levels of TNF-α were decreased in animals treated with 0.5 and 1 mg/kg of DEQX, p < 0.05 and p < 0.001, respectively, compared to positive control.OAQX (5 mg/kg) showed reduced levels of TNF-α (p < 0.001) compared with positive control group.Levels of anti-inflammatory cytokine IL-1β and TNF-α were decreased in the indomethacin treatment compared to positive control (p < 0.001).
Source: Elaborated by the authors.The effect of quinoxaline derivatives on the writhing response in mice is shown in Fig. 8.Both quinoxaline derivatives, DEQX (all doses) and OAQX (5 mg/kg) decreased the writhing response (p < 0.05).In addition, indomethacin significantly decreased the writhing response (p < 0.001).Although a decreasing in the writhing response was observed for both quinoxaline derivatives in different doses, better results were verified for DEQX.
To determine whether cell death induced by DEQX and OAQX was achieved through apoptosis, cells were treated with referred quinoxaline derivatives and stained with Annexin-V-FITC and PI.Using flow cytometry, early and late stages of apoptosis were detected based on the percentage of annexin VFITC-positive cells/PI-negative cells and the percentage of annexin V-FITC-positive/PI-positive cells that were present, respectively (Figs 3, 4 and 5), lower right quadrant data versus top left quadrant data, respectively.
by the authors.
i t i v e c o n t r o l S t a n d a r
by the authors.

Table 1 -
Analgesic activity: hot-plate test and acetic acid writhing reflexNone of DEQX and OAQX (concentrations of 0.5, 1, and 5 mg/kg) presented central analgesic activity (p > 0.05) based on the hot-plate test, as presented in Table1.In contrast, morphine showed central analgesic activity, for 30 minutes Number pain latency time.